title: Complementation of the pha2 yeast mutant suggests functional differences for arogenate dehydratases from Arabidopsis thaliana.
abstract: The final steps of phenylalanine (Phe) biosynthesis in bacteria, fungi and plants can occur via phenylpyruvate or arogenate intermediates. These routes are determined by the presence of prephenate dehydratase (PDT, EC4.2.1.51), which forms phenylpyruvate from prephenate, or arogenate dehydratase (ADT, EC4.2.1.91), which forms phenylalanine directly from arogenate. We compared sequences from select yeast species to those of Arabidopsis thaliana. The in silico analysis showed that plant ADTs and yeast PDTs share many common features allowing them to act as dehydratase/decarboxylases. However, plant and yeast sequences clearly group independently conferring distinct substrate specificities. Complementation of the Saccharomyces cerevisiae pha2 mutant, which lacks PDT activity and cannot grow in the absence of exogenous Phe, was used to test the PDT activity of A. thaliana ADTs in vivo. Previous biochemical characterization showed that all six AtADTs had high catalytic activity with arogenate as a substrate, while AtADT1, AtADT2 and AtADT6 also had limited activity with prephenate. Consistent with these results, the complementation test showed AtADT2 readily recovered the pha2 phenotype after ∼6 days growth at 30 °C, while AtADT1 required ∼13 days to show visible growth. By contrast, AtADT6 (lowest PDT activity) and AtADT3-5 (no PDT activity) were unable to recover the phenotype. These results suggest that only AtADT1 and AtADT2, but not the other four ADTs from Arabidopsis, have functional PDT activity in vivo, showing that there are two functional distinct groups. We hypothesize that plant ADTs have evolved to use the arogenate route for Phe synthesis while keeping some residual PDT activity.
authors.affiliation: Department of Biology, University of Western Ontario, 1151 Richmond Street North, London, Ontario N6A5B7, Canada.
authors.forename: Crystal D
authors.initials: CD
authors.lastname: Bross
authors.forename: Oliver R A
authors.initials: OR
authors.lastname: Corea
authors.forename: Angelo
authors.initials: A
authors.lastname: Kaldis
authors.forename: Rima
authors.initials: R
authors.lastname: Menassa
authors.forename: Mark A
authors.initials: MA
authors.lastname: Bernards
authors.forename: Susanne E
authors.initials: SE
authors.lastname: Kohalmi
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism; D053138:Cyclohexenes / Q000378:metabolism; D005816:Genetic Complementation Test; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010649:Phenylalanine / Q000096:biosynthesis; D011302:Prephenate Dehydratase / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D010649:Phenylalanine; D006836:Hydro-Lyases; C022437:pretyrosine dehydratase; D011302:Prephenate Dehydratase; C005550:prephenic acid